Reperfusion injury is initiated by endothelial dysfunction, which is characterized by increased oxidative stress and limited endothelial-derived nitric oxide (NO) bioavailability, leading to enhanced polymorphonuclear leukocyte (PMN) vascular adherence and infiltration following by blood vessel and organ dysfunction. Therefore, attenuation of endothelial dysfunction may be an effective way to limit reperfusion injury. Endothelial NO synthase (eNOS) plays a central role in normal endothelial function. However, eNOS can shift its product profile from producing NO to superoxide (SO) (eNOS uncoupling) when its essential cofactor, tetrahydrobiopterin (BH4) is oxidized to dihydrobiopterin (BH2) in vitro. Until now, the mechanisms that govern eNOS uncoupling have not been demonstrated by real-time direct measuring oxidative stress during reperfusion in vivo. Recently, we developed a direct measurement of NO and H2O2 (indirect index of SO) release from femoral veins in vivo in a rat femoral artery/vein ischemia/reperfusion (I/R) model using specific H2O2 or NO microsensors. Our preliminary results show that H2O2 is significantly increased in I/R femoral veins compared to non-ischemic (sham) femoral veins in the same animal. Moreover, BH4 or an eNOS inhibitor (e.g., protein kinase C epsilon peptide inhibitor (PKC ?-)) decreased, whereas BH2 or an eNOS enhancer (e.g., PKC ? activator (PKC ?+)) increased H2O2 release during reperfusion. Additionally, BH4 also significantly increased NO release from femoral I/R veins. Furthermore, BH4 or PKC?+ increased, whereas BH2 or PKC ?- decreased NO release in non-ischemic rat aortic segments, respectively. In our ex vivo PMN-induced myocardial I/R injury model, BH4 or PKC ?-, neither BH2 nor PKC ?+ provided cardioprotection when given separately during reperfusion. Based on our preliminary results, we hypothesize that the oxidation of BH4 to BH2 is mainly responsible for eNOS uncoupling during reperfusion, and is the primary cause for opposing effects of PKC ?+/PKC ?- on H2O2/NO release and cardioprotection. To test the hypothesis, we will perform the following specific aims in the presence/absence of BH4, BH2, PKC ?+ or PKC ?- alone or in different combinations: 1) and 2) in vivo measurement of H2O2 or NO release from the femoral I/R vein and sham vein in our femoral I/R model; 3a), 3b) and 4) in vitro NO release measurement from isolated non-ischemic rat aortic segments, ex vivo evaluation of postreperfused cardiac contractile function, heart injury and PMN accumulation. We predict that BH4+PKC ?+ (i.e., eNOS activation), not BH2 +PKC ?+ will significantly decrease oxidative stress and protect heart from reperfusion injury. PUBLIC HEALTH RELEVANCE Characterizing the in vivo and ex vivo mechanism(s) responsible for eNOS uncoupling in reperfusion injury would have a significant impact in identifying more selective treatments, such as BH4 or BH4 combined with PKC ?+, for organ transplants and heart attack patients during reperfusion of blood flow. The clinical impact would be significantly better restoration of organ function resulting in a better quality of life (i.e., aerobic exercise) compared to more sedentary life-style after organ transplantation or heart attacks. [unreadable] [unreadable] [unreadable]